This invention relates to chemically synthesized ribozymes, or enzymatic nucleic acid molecules, antisense oligonucleotides and derivatives thereof.
The following is a brief description of ribozymes and antisense nucleic acids. This summary is not meant to be complete but is provided only for understanding of the invention that follows. This summary is not an admission that all of the work described below is prior art to the claimed invention.
Ribozymes are nucleic acid molecules having an enzymatic activity which is able to repeatedly cleave other separate RNA molecules in a nucleotide base sequence specific manner. Such enzymatic RNA molecules can be targeted to virtually any RNA transcript, and efficient cleavage achieved in vitro. Kim et al., 84 Proc. Natl. Acad. Sci. USA 8788, 1987; Haseloff and Gerlach, 334 Nature 585, 1988; Cech, 260 JAMA 3030, 1988; and Jefferies et al., 17 Nucleic Acids Research 1371, 1989.
Ribozymes act by first binding to a target RNA. Such binding occurs through the target RNA binding portion of a ribozyme which is held in close proximity to an enzymatic portion of the RNA which acts to cleave the target RNA. Thus, the ribozyme first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After a ribozyme has bound and cleaved its RNA target it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.
By xe2x80x9ccomplementarityxe2x80x9d is meant a nucleic acid that can form hydrogen bond(s) with other RNA sequence by either traditional Watson-Crick or other non-traditional types (for example, Hoogsteen type) of base-paired interactions.
Six basic varieties of naturally-occurring enzymatic RNAs are known presently. Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions. Table I summarizes some of the characteristics of these ribozymes. In general, enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.
The enzymatic nature of a ribozyme is advantageous over other technologies, since the effective concentration of ribozyme necessary to effect a therapeutic treatment is lower than that of an antisense oligonucleotide. This advantage reflects the ability of the ribozyme to act enzymatically. Thus, a single ribozyme molecule is able to cleave many molecules of target RNA. In addition, the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base pairing mechanism of binding, but also on the mechanism by which the molecule inhibits the expression of the RNA to which it binds. That is, the inhibition is caused by cleavage of the RNA target and so specificity is defined as the ratio of the rate of cleavage of the targeted RNA over the rate of cleavage of non-targeted RNA. This cleavage mechanism is dependent upon factors additional to those involved in base pairing. Thus, it is thought that the specificity of action of a ribozyme is greater than that of antisense oligonucleotide binding the same RNA site.
By the phrase enzymatic nucleic acid is meant a catalytic modified-nucleotide containing nucleic acid molecule that has complementarity in a substrate binding region to a specified gene target, and also has an enzymatic activity that specifically cleaves RNA or DNA in that target. That is, the enzymatic nucleic acid is able to intramolecularly or intermolecularly cleave RNA or DNA and thereby inactivate a target RNA or DNA molecule. This complementarity functions to allow sufficient hybridization of the enzymatic RNA molecule to the target RNA or DNA to allow the cleavage to occur. 100% Complementarity is preferred, but complementarity as low as 50-75% may also be useful in this invention.
By xe2x80x9cantisense nucleic acidxe2x80x9d is meant a non-enzymatic nucleic acid molecule that binds to another RNA (target RNA) by means of RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm et al., 1993 Nature 365, 566) interactions and alters the activity of the target RNA (for a review see Stein and Cheng, 1993 Science 261, 1004).
By xe2x80x9c2-5A antisense chimeraxe2x80x9d is meant, an antisense oligonucleotide containing a 5xe2x80x2 phosphorylated 2xe2x80x2-5xe2x80x2-linked adenylate residues. These chimeras bind to target RNA in a sequence-specific manner and activate a cellular 2-5A-dependent ribonuclease which in turn cleaves the target RNA (Torrence et al., 1993 Proc. Natl. Acad. Sci. USA 90, 1300).
In preferred embodiments of this invention, the enzymatic nucleic acid molecule is formed in a hammerhead or hairpin motif, but may also be formed in the motif of a hepatitis delta virus, group I intron or RNaseP RNA (in association with an RNA guide sequence) or Neurospora VS RNA. Examples of such hammerhead motifs are described by Rossi et al., 1992, Aids Research and Human Retroviruses 8, 183, of hairpin motifs by Hampel et al., EP0360257, Hampel and Tritz, 1989 Biochemistry 28, 4929, and Hampel et al., 1990 Nucleic Acids Res. 18, 299, and an example of the hepatitis delta virus motif is described by Perrotta and Been, 1992 Biochemistry 31, 16; of the RNaseP motif by Guerrier-Takada et al., 1983 Cell 35, 849, Neurospora VS RNA ribozyme motif is described by Collins (Saville and Collins, 1990 Cell 61, 685-696; Saville and Collins, 1991 Proc. Natl. Acad. Sci. USA 88, 8826-8830; Collins and Olive, 1993 Biochemistry 32, 2795-2799) and of the Group I intron by Cech et al., U.S. Pat. No. 4,987,071. These specific motifs are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule.
The invention provides a method for producing a class of enzymatic cleaving agents which exhibit a high degree of specificity for the RNA of a desired target. The enzymatic nucleic acid molecule is preferably targeted to a highly conserved sequence region of a target such that specific treatment of a disease or condition can be provided with a single enzymatic nucleic acid. Such enzymatic nucleic acid molecules can be delivered exogenously to specific cells as required. In the preferred hammerhead motif the small size (less than 60 nucleotides, preferably between 30-40 nucleotides in length) of the molecule allows the cost of treatment to be reduced compared to other ribozyme motifs.
Synthesis of nucleic acids greater than 100 nucleotides in length is difficult using automated methods, and the therapeutic cost of such molecules is prohibitive. In this invention, small enzymatic nucleic acid motifs (e.g., of the hammerhead structure) are used for exogenous delivery. The simple structure of these molecules increases the ability of the enzymatic nucleic acid to invade targeted regions of the mRNA structure. Unlike the situation when the hammerhead structure is included within longer transcripts, there are no non-enzymatic nucleic acid flanking sequences to interfere with correct folding of the enzymatic nucleic acid structure or with complementary regions.
Eckstein et al., International Publication No. WO 92/07065, Perrault et al. Nature 1990, 344, 565-568, Pieken, W. et al. Science 1991, 253, 314-317, Usman, N.; Cedergren, R. J. Trends in Biochem. Sci. 1992, 17, 334-339, Usman, N. et al. International Publication No. WO 93/15187 and Sproat, B. U.S. Pat. No. 5,334,711 describe various chemical modifications that can be made to the sugar moieties of enzymatic RNA molecules. All these publications are hereby incorporated by reference herein.
Medina et al., 1988 Tetrahedron Letters 29, 3773, describe a method to convert alcohols to methylthiomethyl ethers.
Matteucci et al., 1990 Tetrahedron Letters, 31, 2385, report the synthesis of 3xe2x80x2-5xe2x80x2-methylene bond via a methylthiomethyl precursor.
Veeneman et al., 1990 Recl. Trav. Chim. Pays-Bas 109, 449, report the synthesis of 3xe2x80x2-O-methylthiomethyl deoxynucleoside during the synthesis of a dimer containing 3xe2x80x2-5xe2x80x2-methylene bond.
Jones et al., 1993 J. Org. Chem. 58, 2983, report the use of 3xe2x80x2-O-methylthiomethyl deoxynucleoside to synthesize a dimer containing a 3xe2x80x2-thioformacetal internucleoside linkages. The paper also describes a method to synthesize phosphoramidites for DNA synthesis.
Zavgorodny et al., 1991 Tetrahedron Letters 32, 7593, describe a method to synthesize a nucleoside containing methylthiomethyl modification.
This invention relates to the incorporation of 2xe2x80x2-O-R3-thio-R3 and/or 2xe2x80x2-C-R3-thio-R3 nucleotides or non-nucleotides into nucleic acids, which are particularly useful for enzymatic cleavage of RNA or single-stranded DNA, and also as antisense oligonucleotides. As used herein, each R3 is independently a compound selected from a group consisting of alkyl, alkenyl, alkynyl, aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester.
As the term is used in this application, 2xe2x80x2-O-R3-thio-R3 and/or 2xe2x80x2-C-R3-thio-R3 nucleotide or non-nucleotide-containing enzymatic nucleic acids are catalytic nucleic molecules that contain 2xe2x80x2-O -R3-thio-R3 and/or 2xe2x80x2-C -R3-thio-R3 nucleotide or non-nucleotide components replacing one or more bases or regions including, but not limited to, those bases in double stranded stems, single stranded xe2x80x9ccatalytic corexe2x80x9d sequences, single-stranded loops or single-stranded recognition sequences. These molecules are able to cleave (preferably, repeatedly cleave) separate RNA or DNA molecules in a nucleotide base sequence specific manner. Such catalytic nucleic acids can also act to cleave intramolecularly if that is desired. Such enzymatic molecules can be targeted to virtually any RNA transcript.
Also within the invention are 2xe2x80x2-O-R3-thio-R3 and/or 2xe2x80x2-C-R3-thio-R3 nucleotides or non-nucleotides which may be present in enzymatic nucleic acid or in antisense oligonucleotides or 2-5A antisense chimera. Such nucleotides or non-nucleotides are useful since they enhance the activity of the antisense or enzymatic molecule. The invention also relates to novel intermediates useful in the synthesis of such nucleotides or non-nucleotides and oligonucleotides (examples of which are shown in the Figures), and to methods for their synthesis.
Thus, in a first aspect, the invention features 2xe2x80x2-O- -R3-thio-R3 nucleosides or non-nucleosides, that is a nucleoside or non-nucleosides having at the 2xe2x80x2-position on the sugar molecule a 2xe2x80x2-O -R3-thio-R3 moiety. In a related aspect, the invention also features 2xe2x80x2-O -R3-thio-R3 nucleotides or non-nucleotides. That is, the invention preferably includes those nucleotides of non-nucleotides having 2xe2x80x2 substitutions as noted above useful for making enzymatic nucleic acids or antisense molecules that are not described by the art discussed above.
The term non-nucleotide refers to any group or compound which can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity. The group or compound is abasic in that it does not contain a commonly recognized nucleotide base, such as adenine, guanine, cytosine, uracil or thymine. It may have substitutions for a 2xe2x80x2 or 3xe2x80x2 H or OH as described in the art. See Eckstein et al. and Usman et al., supra.
The term nucleotide refers to the regular nucleotides (A, U, G, T and C) and modified nucleotides such as 6-methyl U, inosine, 5-methyl C and others. Specifically, the term xe2x80x9cnucleotidexe2x80x9d is used as recognized in the art to include natural bases, and modified bases well known in the art. Such bases are generally located at the 1xe2x80x2 position of a sugar moiety. The term xe2x80x9cnon-nucleotidexe2x80x9d as used herein to encompass sugar moieties lacking a base or having other chemical groups in place of a base at the 1xe2x80x2 position. Such molecules generally include those having the general formula: 
wherein, R1 represents 2xe2x80x2-O -R3-thio-R3 or 2xe2x80x2-C -R3-thio-R3; X represents a base of H; Y represents a phosphorus-containing group; and R2 represents H, DMT or a phosphorus-containing group.
Phosphorus-containing group is generally a phosphate, thiophosphate, H-phosphonate, methylphosphonate, phosphoramidite or other modified group known in the art.
In a second aspect, the invention features 2xe2x80x2-C -R3-thio-R3 nucleosides or non-nucleosides, that is a nucleotide or a non-nucleotide residue having at the 2xe2x80x2-position on the sugar molecule a 2xe2x80x2-C-R3-thio-R3 moiety. In a related aspect, the invention also features 2xe2x80x2-C-R3-thio-R3 nucleotides of non-nucleotides. That is, the invention preferably includes all those 2xe2x80x2 modified nucleotides or non-nucleotides useful for making enzymatic nucleic acids or antisense molecules as described above that are not described by the art discussed above.
Specifically, an xe2x80x9calkylxe2x80x9d group refers to a saturated aliphatic hydrocarbon, including straight-chain, branched-chain, and cyclic alkyl groups. Preferably, the alkyl group has 1 to 12 carbons. More preferably it is a lower alkyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkyl group may be substituted or unsubstituted. Whyen substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, xe2x95x90O, xe2x95x90S, NO2 or N(CH3)2, amino, or SH. The term alkenyl refers to unsaturated hydrocarbon groups containing at least one carbon-carbon double bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkeny group has 1 to 12 carbons. More preferably it is a lower alkenyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkenyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, xe2x95x90O, xe2x95x90S, NO2, halogen, N(CH3)2, amino, or SH. The term alkynyl refers to an unsaturated hydrocarbon group containing at least one carbon-carbon triple bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkynyl group has 1 to 12 carbons. More preferably it is a lower alkynyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkynyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, xe2x95x90O, xe2x95x90S, NO2 or N(CH3)2, amino or SH.
An xe2x80x9carylxe2x80x9d group refers to an aromatic group which has at least one ring having a conjugated xcfx80 electron system and includes carbocyclic aryl, heterocyclic aryland biaryl groups, all of which may be optionally substituted. The preferred substituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An xe2x80x9calkylarylxe2x80x9d group refers to an alkyl group (as described above) covalently joined to an aryl group (as described above. Carbocyclic aryl groups are groups wherein the ring atoms on the aromatic ring are all carbon atoms. The carbon atoms are optionally substituted. Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted. An xe2x80x9camidexe2x80x9d refers to an xe2x80x94C(O)xe2x80x94NHxe2x80x94R, where R is either alkyl, aryl, alkylaryl or hydrogen. An xe2x80x9cesterxe2x80x9d refers to an xe2x80x94C(O)xe2x80x94ORxe2x80x2, where R is either alkyl, aryl, alkylaryl or hydrogen.
In other aspects, also related to those discussed above, the invention features oligonucleotides having one or more 2xe2x80x2-O -R3-thio-R3 and/or 2-xe2x80x2-C -R3-thio-R3 nucleotides or non-nucleotides; e.g. enzymatic nucleic acids having 2xe2x80x2-O-R3-thio-R3 and/or 2xe2x80x2-C-R3-thio-R3 nucleotides of non-nucleotides; and a method for producing an enzymatic nucleic acid molecule having enhanced activity to cleave an RNA or single-stranded DNA molecule, by forming the enzymatic molecule with at least one nucleotide or a non-nucleotide moiety having at its 2xe2x80x2-position a 2xe2x80x2-O-R3-thio-R3 and/or 2xe2x80x2-C-R3-thio-R3 group.
In other related aspects, the invention 2xe2x80x2-O-R3-thio-R3 and/or 2xe2x80x2-C-R3-thio-R3 nucleotide triphosphates. These triphosphates can be used in standard protocols to form useful oligonucleotides of this invention.
The 2xe2x80x2-O -R3-thio-R3 and/or 2xe2x80x2-C -R3-thio-R3 derivatives of this invention provide enhanced activity and stability to the oligonucleotides containing them.
In yet another preferred embodiment, the invention features oligonucleotides having one or more 2xe2x80x2-O-R3-thio-R3 and/or 2xe2x80x2C -R3-thio-R3 abasic (non-nucleotide) moieties. For example, enzymatic nucleic acids having a 2xe2x80x2-O -R3-thio-R3 and/or 2xe2x80x2-C -R3-thio-R3 abasic moeity; and a method for producing an enxymatic nucleic acid molecule having enhanced activity to cleave an RNA or single-stranded DNA molecule, by forming the enzymatic molecule with at least one position having at its 2xe2x80x2-position an 2xe2x80x2-O -R3-thio-R3 and/or 2xe2x80x2-C -R3-thio-R3.
In related embodiments, the invention features enzymatic nucleic acids containing one or more 2xe2x80x2-O-R3-thio-R3 and/or 2xe2x80x2-C-R3-thio-R3 substitutions either in the enzymatic portion, substrate binding portion or both, as long as the catalytic activity of the ribozyme is not significantly decreased.
By xe2x80x9cenzymatic portionxe2x80x9d is meant that part of the ribozyme essential for cleavage of an RNA substrate.
By xe2x80x9csubstrate binding armxe2x80x9d is meant that portion of a ribozyme which is complementary to (i.e., able to base-pair with) a portion of its substrate. Generally, such complementarity is 100%, but can be less if desired. For example, as few as 10 bases out of 14 may be base-paired. Such arms are shown generally in FIGS. 1-3 as discussed below. That is, these arms contain sequences within a ribozyme which are intended to bring ribozyme and target RNA together through complementary base-pairing interactions; e.g., ribozyme sequences within stems I and III of a standard hammerhead ribozyme make up the substrate-binding domain (see FIG. 1).
In yet another preferred embodiment, the invention features the use of 2xe2x80x2-O-alkylthioalkyl moieties as protecting groups for 2xe2x80x2-hydroxyl positions of ribofuranose during nucleic acid synthesis.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.